Project Summary/Abstract Inspired Dr. Robert A. Wineberg?s belief that a landmark drug will be one that attacks the pathways that keep cancer cells alive via a multi-strategic mechanism, a multimodal anticancer drug strategy has been designed to attack cellular iron. Iron is crucial to the growth and proliferation of cancer cells and the onset of metastasis. Cancer cells overexpress proteins responsible for iron uptake and iron functionalization in order to meet their higher metabolic demand relative to normal cells. In this proposal, titanium(IV) compounds made with iron chelators as ligands (Ti(IV) IC compounds) are designed to synergize the antiproliferative/cytotoxic properties of iron chelators and Ti(IV), a redox-inert Fe(III) biomimic, to suppress the bioavailability and alter the functionality of the cellular labile iron pool via both Ti(IV) for Fe(III) transmetalation and direct Fe(II) coordination. The labile Fe(II) and Fe(III) is converted into redox active cytotoxic species that together with released Ti(IV) inhibit the iron-dependent enzyme ribonucleotide reductase (RNR). The iron chelator component of the Ti(IV) IC compounds will consist of two chelating moieties, deferasirox and triapine. Deferasirox, an FDA approved drug to treat iron overload diseases, forms very stable Ti(IV) compounds with an enhanced specificity for Fe(III) binding. In the cellular environment, labile Fe(III) induces the dissociation of Ti(deferasirox)2 to form the Fe(deferasirox)2 complex, which is functionally inert. Triapine binds both Fe(II) and Fe(III) and is in various anticancer clinical trials. Fe coordination by triapine produces redox active Fe species, which have the capacity to generate excessive reactive oxygen species and to reductively inactivate the R2 domain of the RNR enzyme at its diiron activation site. As the only protein responsible for producing deoxyribonucleotides, the building blocks for DNA replication, RNR inhibition has been a major anticancer drug target. In this project, deferasirox and triapine will be coupled both by direct conjugation and through Ti(IV) coordination to generate Ti(IV) IC compounds. It is hypothesized that these compounds will exhibit excellent potency against cancer cells because of their capacity to attack and weaponize both the Fe(II) and Fe(III) species of the labile pool to facilitate reductive inactivation and Ti(IV) inhibition of the RNR enzyme. Mechanistic studies will be performed to understand the contribution of the chelator moieties and Ti(IV) to the cytotoxic and antiproliferative properties of the compounds. It will be determined whether a correlation exists between the generation of redox-active Fe species by triapine and deferasirox binding of the metal and the induction of apoptosis. Metal competition studies and electron paramagnetic resonance analyses with recombinantly expressed R2 RNR will help to determine whether Ti(IV) can bind at its diiron site, inhibit Fe binding and, due to its redox inertness, prevent activation of this site. These studies have the potential to reveal a novel antiproliferative property of Ti(IV). The therapeutic index of these compounds and their ability to operate via the desired multimodal approach will be used as gauges to determine the appropriate Ti(IV) IC compound composition that optimizes activity within cells.